Molecular vector for the genophenotypic characterisation of v3 sequences of hiv-1 gp120 and process for preparation thereof

ABSTRACT

Recombinant vector essentially consisting of: a) a plasmid component able to carry out exactly and efficiently vector replication in bacteria and viral insert expression in eukaryotic cells; b) an HIV component characterised in that the V3 portion sequence of env gene has been deleted and substituted by Nrul enzyme restriction site. In addition the invention relates to a method for the genophenotypic evaluation of HIV genomic sequences.

The present invention relates to a molecular vector for thegenophenotypic characterisation of V3 sequences of HIV-1 gp120 andprocess for preparation thereof.

It is known that human immunodeficiency virus, HIV, presents on theoutside envelope surface proteins, like gp120 and gp41, which play a keyrole during the initial phase of virus infection.

Entry phase of HIV virus in the host cell is a complex processdeveloping in various steps, including gp120 surface glycoproteinbinding to CD4 cell receptor of host cell, gp120 conformationalmodifications resulting in gp41 exposure and subsequent co-receptorselection before the starting of the fusion-type activity by gp41glycoprotein. The latter, in fact, promotes the fusion of the lipidenvelope and plasmatic membrane of the host cell to obtain a continuousunique membrane resulting in the introduction of the viral nucleoproteincore into cytoplasm of the host cell.

CD4 cell receptors are present on two types of hematic cells, namely CD4lymphocytes (T helper lymphocytes) and monocytes. Various studiesidentified as co-receptors various surface molecules, named CCR5 andCXCR4, physiologically assigned to interact with some chemokines namedRANTES (Regulated-Activations Normal T Expressed Secreted), MIP1-α,MIP1-β (Macrophage Inhibitory Protein) (Moore, J. P., A. Trkola and T.Dragic, 1997. Co-receptors for HIV-1 entry. Curr. Opin. Immunol. 9,551-562). These co-receptors are both on T lymphocytes and macrophages.

gp120 variable sequences and particularly the 3 variable region (V3)play a key role during the very early steps of the HIV infectionprocess, particularly for the binding and binding selection to thereceptor and various co-receptors, respectively. Further V3 region ischaracterised by a remarkable inter and intra-host genomic diversity andis exposed to the host selection activities (Cocchi, F. et al., 1996,Nat. Med. 2, 1244-1247).

Accordingly it is known that during the natural history of HIV infectionthe viral population undergoes a non homogeneous genetic evolutionresulting in viral variants (quasispecies) and characterising thedisease progress.

During the genetic evolution the modifications involving the envstructural gene are particularly important. The latter encodes for apolypeptide precursor, gp160, which subsequently is cleaved by viralproteases generating gp120 and gp41 glycoproteins.

Modifications of env gene and therefore V3 regions of gp120 affect thebinding to the various co-receptors and develop in detectableparallelism with the disease progress.

Finally the HIV entry step, in addition to represent the main step ofthe viral replication to be overcome by main tests for activeimmunization, currently is also an attracting target for the developmentof new compounds with antiviral activity comprising both inhibitors ofthe bond between V3 region of HIV gp120 and CCR5 and CXCR4 co-receptorsand of virus fusion with the host cell, respectively.

Due to these main reasons techniques and procedures suitable to studyone or more aspects related to this step are important both as tools toinvestigate the infection from pathogenic point on view and potentialdiagnostic methodologies.

In the recent years individual molecular recombinant clones weredeveloped to evaluate the functionality of partial deletion mutants ofenv gene sequences. In most cases evaluation studies of individualclones or chimeric viruses for research purposes were carried out.(Isaka, Y. et al., 1999, 264; 237-43; Chan, S. Y et al., J Virol. 73:2350-8). In some cases procedures involving “complementation” of viralsequences present in two different subsequently co-transfected vectors,wherein one thereof carries the viral skeleton lacking for a sequenceand the other carries the lacking sequence from heterologous derivation,were used; this approach, although interesting when used for researchpurposes, experiences inquiring and, at the end, inefficient whenapplied for diagnostic aspect.

From the above description it is apparent the need to have theavailability of new tools and methodologies for the study of thestructures and mechanisms involved in the entry step of the HIVinfection.

The authors of the present invention therefore provided a method for thegenophenotypic evaluation of genomic sequences and correspondingstructures involved in the virus entry step of in vivo selected or invitro generated variants.

Within the scope of the invention, therefore, a molecular vector to beadvantageously used for the evaluation of the recombinant phenotype ofthe V3 region and corresponding analytical methodology were provided.

Molecular vector includes the HIV genome, modified by the deletion ofthe sequence encoding for V3 region and insertion of a new restrictionsite. This vector allows an easy insertion of exogenous V3 sequences andthe production of recombinant virus (chimeric) after the transfection ofthe same vector in competent cells.

The inventive method allows easy and rapid cloning of V3 sequences in amolecular vector to be carried out and, after transfection, recombinantlarge scale viruses to be obtained and studied and, concurrently, theobtained clones to be handled easily by mutagenesis.

Said vector has been constructed and peculiar feature thereof is theavailability for a routine work aiming at the evaluation of individualclones of the viral V3 sequence. It is possible by virtue of specificmodifications carried out on the backbone. These modifications, designedin a such way not to modify the frameshift of the translation after theexpression of the viral sequences occurring in the transfected cells,concern the region wherein the insertion for the cloning of theheterologous V3 sequence is carried out and the specific usedrestriction site.

A further advantage is represented by the easiness of the cloningimproving the efficiency of a method suitable to a routine work.

The validation by means of reference strains has confirmed that theobtained recombinant virus maintains, in respect of the use ofco-receptors, the characteristics of the strain from which the V3sequence has been obtained.

An interesting feature for studies both about pathogenesis andresistance of drugs to inhibitors of co-receptor use is the possibilityof obtaining mutants of V3 region in a simple, direct and rapid way andevaluating immediately phenotypic characteristics thereof.

It is therefore a specific object of the present invention a recombinantvector essentially consisting of: a) a plasmid component able to carryout exactly and efficiently vector replication in bacteria and viralinsert expression in eukaryotic cells, respectively; b) an HIV componentcharacterised in that the V3 portion sequence of env gene has beendeleted and substituted by Nrul enzyme restriction site.

It is a further object of the present invention a method forgenophenotypic evaluation of HIV genome sequences and correspondingstructures involved in virus entry step of in vivo selected or in vitrogenerated variants, comprising essentially the following steps: 1)amplification of the sequence encoding for V3 region from a sample; 2)cloning of the amplification product in the inventive vector in order toobtain a recombinant vector, 3) transfection of the recombinant vectorin competent cells like, for example, 293T line cells, able to carry outHIV replication; 4) collection and purification of the obtainedrecombinant HIV virus; 5) infection of cell lines by obtainedrecombinant HIV viruses expressing different receptors and/orco-receptors able to bind HIV V3 sequence.

Preferably the amplification of the sequence encoding for V3 region froma sample is carried out by using following primers: V3s5′-TACAGCTGAAGTAATCTGTAGAAAT; V3as 5′-TATTCCATTTTGCTCTACTAA.

Among receptors and co-receptors expressed by infectible cell linesthere are CD4 receptors and CCR5 and CXCR4 co-receptors.

The present invention will be described now, by way of illustration butnot limitation, according to preferred embodiments thereof, particularlywith reference to the enclosed drawings, wherein:

FIG. 1 shows the construction of the pNL-ΔV3 suitable to be used toobtain recombinant viruses for V3 sequence.

FIG. 2 shows the procedure used in the study for the evaluation of theviral phenotype from V3 sequences obtained from clinical sample ofinfected patients.

FIG. 3 shows the results of the general validation of the procedure.

EXAMPLE 1 Construction of pNL-ΔV3 Vector

Vector suitable to obtain recombinant viruses of V3 sequences wasobtained by using the procedure described in FIG. 1.

HIV specific viral insert, contained in pNL4-3 vector (NIBSC-MCRCentralised Facility for AIDS Reagents; cat N. 2006), firstly wastranslated, by cutting and subsequent cloning, into pCR-Script plasmid(cat. Invitrogen). Successively the HIV env gene fragment limited byNhel and Stul restriction sites and including V3 sequence was modifiedand amplified by PCR-directed mutagenesis.

Following two primers were used: NheI-NruI primer5′-GCgctagcTATCTGTTTTAAAGtcgcaTTATGGTTTTAGC; StuI primer5′-cctGTCCAAAGGTATCCTTTGA

First primer included a deletion of V3 sequence and, concurrently,generated a new restriction site of Nrul enzyme. The latter, whosesequence is AGCGCT, is not contained in the HIV genome and allowsheterologous V3 sequences to be easily inserted.

The vector, named NLmodΔV3, was used for cloning of heterologous V3sequences in Nrul site.

EXAMPLE 2

Methodology for the study of phenotypic characteristics conferred by invivo selected V3 sequences.

Molecular vector obtained according to example 1 was used in amethodology for the study of phenotypic viral characteristics conferredby in vivo selected V3 sequences from infected patients.

The methodology is showed in FIG. 2 and is characterised by thefollowing steps: amplification of V3 sequence from clinical sample usingfollowing primers: V3s 5′-TACAGCTGAAGTAATCTGTAGAAAT; V3as5′-TATTCCATTTTGCTCTACTAA.

Sequences are just outside V3 region and designed to allow cloning ofthe amplified in Nrul site.

Subsequent step is cloning of the amplification product within the newvector followed by transfection into competent cells, collection andpurification of freshly produced recombinant virus (common backbone andclone specific V3 sequence), infection of different cell lines andpositive CD4 cells expressing a single co-receptor.

Clonal V3 sequences, obtained by PCR carried out, alternatively, onplasma purified RNA or peripheral lymphocyte extracted DNA, wereinserted into NLmodΔV3 vector by using Nrul restriction site.Successively competent bacterial cells were transformed and the plasmidwas purified. Cloned V3 sequence was then amplified starting from thevector and sequenced. Complete vector was used to transfect 293T linecells (National Institue of Health—AIDS Research and Reference Reagent;cat N. 103) by lipofectine transfection. Infecting virus, produced fromcells transfected by complete vector, was collected from cellsupernatant and used to infect primary lymphocytes, MT2 cells (NIBSC—MCRCentralised Facility for AIDS Reagents; N. ARP014), PM1 cells (lymphoidT lines) and, particularly, U87 cells (NIBSC—MCR Centralised Facilityfor AIDS Reagents; N. ARP069-073) which express chemokine receptors. U87cells are genetically modified in order to express individually anyoneof HIV receptors used during the cell entry step.

Chemokine receptors, named CCR5 and CXCR4, are the most used by virus.HIV can be able to use CCR5 or CXCR4. There are viral strains able touse both receptors (dual-tropic strains).

It is apparent that the selection of binding to various co-receptors canin vivo affect the tropism, i.e. the ability of the virus to infect aspecific cell or tissue, because receptors are differently distributedon the surface of infectible cells.

U87 cells which express individual receptors are therefore an excellentmodel to evaluate the viral phenotype with respect to the initial stepof the infecting cycle. When it is used a viral skeleton on which theshort V3 sequence only varies, the detected phenotypic modificationsresult from the sequence itself. In other words a biological modelsuitable to evaluate the significance of V3 sequence modifications willbe available.

EXAMPLE 3 Procedure Validation

The procedure validation was carried out by cloning into the new vectorV3 sequences of reference viral clones having known CCR5 and CXCR4tropism and dual-tropic CCR5-CXCR4 clones. Particularly V3 sequences ofviral NL4-3 clones were used, known as virus able to use AD8, CXCR4receptor (Theodore Ts, et al., 1996, AIDS Res Hum Retrovir 12, 191-194)which uses dual-tropic CCR5 and SF2 (NIBSC-MCR Centralised Facility forAIDS Reagents; N ARP112.1) receptor, i.e. it is able to use either CCR5or CXCR4 receptor.

Reported sequences were PCR amplified and cloned into NLmodΔV3 vector byusing Nrul restriction site. According to the procedure described inFIG. 2 a complete extracellular virus was obtained, which was then usedto infect various lymphoid lines and U87 cells which express CCR5 orCXCR4 on the surface. X4 or R5 terms mean virus able to usealternatively CXCR4 or CCR5 receptors.

Obtained results confirmed that recombinant viruses, differing for V3region only, keep the original virus tropism.

Viral clones used for validation tests were produced by the authors(Menzo, S. et al., 1998, 12: 985-97; P. Bagnarelli et al., Journal ofVirology 73, 3764-3777, 1999).

1. Recombinant vector essentially consisting of: a) a plasmid componentable to carry out exactly and efficiently vector replication in bacteriaand viral insert expression in eukaryotic cells; b) an HIV componentcharacterised in that the V3 portion sequence of env gene has beendeleted and substituted by Nrul enzyme restriction site.
 2. Method forgenophenotypic evaluation of HIV genome sequences and correspondingstructures involved during the virus entry step of in vivo selected orin vitro generated variants, comprising essentially the followingsteps: 1) amplification of the sequence encoding for V3 region from asample; 2) cloning of the amplification product in the vector accordingto claim 1 in order to obtain a recombinant vector; 3) transfection ofthe recombinant vector in competent cells like able to carry out HIVreplication; 4) collection and purification of the obtained recombinantHIV virus; 5) infection of cell lines by obtained recombinant HIVviruses which express different receptors and/or co-receptors able tobind HIV V3 sequence.
 3. Method according to claim 2 wherein theamplification of the sequence encoding for V3 region from a sampleoccurs by using the following primers: V3s 5′-TACAGCTGAAGTAATCTGTAGAAAT(SEQ ID NO.1) V3as 5′-TATTCCATTTTGCTCTACTAA. (SEQ ID NO.2)


4. Method according to claim 2 wherein the receptors are CD4s and theco-receptors are CCR5s and CXCR4s.
 5. Method according to claim 2wherein the competent cells are 293T line cells.